| 斑马鱼性腺组织的原代细胞培养技术的建立 |
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| 引用本文： | 董丹丹,孙燕侠,郭华荣.斑马鱼性腺组织的原代细胞培养技术的建立[J].安徽农业科学,2016,44(15):130-134. |
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| 作者姓名： | 董丹丹 孙燕侠 郭华荣 |
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| 作者单位： | 中国海洋大学海洋生命学院,山东青岛,266003;中国海洋大学海洋生命学院,山东青岛,266003;中国海洋大学海洋生命学院,山东青岛,266003 |
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| 基金项目： | 国家自然科学基金项目(31472274，31172391)；海洋经济创新发展区域示范项目(12 PYY001 SF08)；中国海洋大学本科生SRDP项目。 |
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| 摘 要： | 目的]建立斑马鱼卵巢和精巢组织的原代细胞培养技术,获得原代和传代培养细胞单层。方法]采用组织块贴壁法进行斑马鱼卵巢和精巢组织的原代细胞培养,采用0.25%胰蛋白酶消化法进行传代培养。结果]斑马鱼性腺组织的原代细胞培养条件为:最适培养基为DMEM/F12(p H 7.0~7.2),培养温度为24℃,在添加20 ng/m L表皮生长因子(EGF)、20 ng/m L碱性成纤维细胞生长因子(b FGF)、0.5 mmol/Lβ-巯基乙醇和15%胎牛血清时更有利于细胞的存活。其中,卵巢的组织块接种3~4 d后,上皮样细胞首先开始迁出,迁出的上皮样细胞生长5 d后,逐渐凋亡;此后,成纤维样细胞和另一种上皮样细胞开始大量迁出,10 d后可生长汇合成80%的原代细胞单层;可成功传代培养1次,但传代后的上皮样细胞不贴壁,很快死亡,而传代后的成纤维样细胞可贴壁生长,但基本不分裂,健康存活7 d后开始逐渐凋亡。精巢组织接种5 d后,上皮样细胞和成纤维样细胞同时开始迁出,培养16 d后逐渐凋亡,仅得到60%原代培养细胞单层。结论]初步建立了斑马鱼性腺组织的原代细胞培养条件,并将卵巢组织块迁出的成纤维样细胞成功传代1次。
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| 关 键 词： | 斑马鱼 性腺组织 原代细胞培养 传代培养 |
Development of Primary Cell Cu lture from the Gonad Tissues of Zebrafish |
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| DONG Dan-dan,SUN Yan-xia,GUO Hua-rong.Development of Primary Cell Cu lture from the Gonad Tissues of Zebrafish[J].Journal of Anhui Agricultural Sciences,2016,44(15):130-134. |
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| Authors: | DONG Dan-dan SUN Yan-xia GUO Hua-rong |
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| Abstract: | Objective] To develop a primary cell culture system for the cells from ovary and testis tissues of zebrafish and to obtain the corre -sponding cell monolayer of both primary culture and subculture.Method]Primary cell cultures were derived using the explants culture method. The primary cells were sub-cultured by 0.25% trypsin digestion.Result] The zebrafish gonad-derived cells were cultured in DMEM/F12 (pH 7.0-7.2) at an optimal temperature of 24 ℃.The cells had better growth and survivor when the medium mentioned above was supplemented with 20 ng/mL epidermal growth factor (EGF),20 ng/mL basic fibroblast factor (bFGF),0.5 mmol/Lβ-mercaptoethanol and 15%fetal bovine serum (FBS).For ovary tissues,epithelium-like cells first migrated from the explants at 3~4 days after seeding.These epithelium-like cells could only survive for about 5 days in vitro,and then tended to apoptosis.However, active migration of fibroblast-like cells and another kind of epitheli-um-like cells were then observed and a primary cell monolayer with 80%confluence could be formed within10 days.Then the primary cell mono-layer could be successfully sub-cultured once.The epithelium-like cells can’t re-attach and die soon after subculture.However,the re-attached fi-broblast-like cells could healthily survive for another 7 days and then tend to apoptosis.No obvious cell division was observed in both the primary and subculture cells.For testis tissues,both epithelium-like cells and fibroblast-like cells migrated from the explants at 5 days after seeding.At 16 days after seeding,a primary cell monolayer with 60%confluence was obtained and then tended to apoptosis.Conclusion] This study has estab-lished a primary cell culture system suitable for the gonad tissues of zebrafish ,and the fibroblast-like cell monolayer derived from the ovary tissues has been successfully sub-cultured for one time. |
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| Keywords: | Zebrafish Gonad tissue Primary cell culture Subculture |
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